Method for the synthesis of tryptophan analogs in aqueous solvents at reduced temperatures
a technology of aqueous solvent and tryptophan analog, which is applied in the direction of lyases, carbon-oxygen lyases, enzymology, etc., can solve the problems of multiple steps, the need to pre-synthesize the water-sensitive electrophilic, etc., and achieve the effect of improving activity
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Benefits of technology
Problems solved by technology
Method used
Image
Examples
example 1
neering for Improved Synthesis of Tryptophan Variants
[0160]General Experimental Methods.
[0161]Chemicals and reagents were purchased from commercial sources and used without further purification. The proton NMR spectrum was recorded on a Bruker 400 MHz (100 MHz) spectrometer equipped with a cryogenic probe. Proton chemical shifts are reported in ppm (δ) relative to tetramethylsilane and calibrated using the residual solvent resonance (DMSO, δ 2.50 ppm). The NMR spectrum was recorded at ambient temperature (about 25° C.). Preparative reversed-phase chromatography was performed on a Biotage Isolera One purification system, using C18 silica as the stationary phase, with CH3OH as the strong solvent and H2O (0.1% HCl by weight) as the weak solvent. Liquid chromatography / mass spectrometry (LCMS) was performed on an Agilent 1290 UPLC-LCMS equipped with a C-18 silica column (1.8 μm, 2.1×50 mm) using CH3CN / H2O (0.1% acetic acid by volume): 5% to 95% CH3CN over 4 min; 1 mL / min. The optical pur...
example 2
le Production of 4-Cyanotryptophan
[0198]The enzyme Tm9D8* was prepared in four 250 mL TBamp expression cultures according to the procedure described above. The cell pellet (16.6 grams) was lysed by resuspension in 66 mL of 50 mM KPi buffer that contained 7.6 mg PLP, 72 mg HEWL, and 6.7 mg DNase. BugBuster® (7.8 mL, 10× concentration) was added, and then the suspension was shaken at 230 RPM and 37° C. for 15 minutes. The suspension was subjected to centrifugation at 4,500 g and 4° C. (5 minutes) and then immersed in a water bath at 75° C. After 30 minutes, the heat-treated lysate was cooled on ice, then subjected to centrifugation at 15,000 g and 4° C. (15 minutes). In a 500-mL Erlenmeyer flask, 4-cyanoindole (1.0 g, 7.0 mmol) and serine (810 mg, 7.7 mmol) were suspended in DMSO (35 mL) and 50 mM KPi buffer (750 mL). Heat-treated lysate (75 mL) was added, then the reaction mixture was incubated at 40° C. and 60 rpm. After 65 hours, the reaction mixture was cooled on ice for 90 minute...
example 3
TrpB Activity / Temperature Profile
[0200]The activity of Tm9D8* at 30° C. was tested with a set of 5-substituted indole analogues (5-OH—, 5-OMe-, 5-Br—, 5-CN-indole). Each reaction was prepared by charging a 2 mL glass HPLC vial with 10 μL of a 0.4 M solution of the indole analogue in DMSO, followed by the addition of 190 μL of a solution containing enzyme, serine, and potassium phosphate buffer (50 mM, pH 8.0). This gave final concentrations of 4 μM enzyme (0.02 mol %, 5000 maximum turnovers), 20 mM serine, 20 mM indole analogue, and 5% DMSO in a total volume of 200 μL. The reactions were then incubated in a 30° C. water bath for 6 hours and then worked up with 800 L of a 1:1 1 M aq. HCl / CH3CN mixture and vortexed. The reactions were transferred to microcentrifuge tubes and subjected to centrifugation at >10,000 g for 10 min and analyzed via LCMS. Product yield was calculated by comparing the integrations of the product and nucleophile absorbance peaks at 254 nm.
RxnApprox-TempAreaAre...
PUM
| Property | Measurement | Unit |
|---|---|---|
| Temperature | aaaaa | aaaaa |
| Temperature | aaaaa | aaaaa |
| Fraction | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to View More